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Interaction of a bisquaternary ammonium compound with the peripheral organophosphorus (POP) site on acetylcholinesterase
Neurochem. Int. Vol. 9, No. 2, pp. 323 328, 1986 Printed in Great Britain
0197-0186/86 $3.00 +0.00 Pergamon Journals Ltd
INTERACTION OF A BISQUATERNARY A M M O N I U M C O M P O U N D WITH THE PERIPHERAL ORGANOPHOSPHORUS (POP) SITE ON ACETYLCHOLINESTERASE ALAIN FRIBOULET*, DANIf/LE GOUDOU and FRAN(~OIS RIEGER Unit6 de Biologie et Pathologie Neuromusculaires INSERM U 153, 17 rue du Fer-fi-Moulin, 75005 Paris, France (Received 8 August 1985; accepted 24 February 1986) Abstraet--O-ethyl-S (2 diisopropylaminoethyl) methyl phosphorothiolate (MPT) is an active site-directed inhibitor of acetylcholinesterase (ACHE). The inhibition of mouse muscle AChE by MPT as well as the inhibition of its individual molecular forms do not proceed as simple irreversible bimolecular reactions. The insolubilization of AChE into a semisolid matrix allows to characterize, after dialysis of all unbound ligand, a partially reversible phase of the inhibition by MPT. These results can be explained in terms of two different modes of inhibition by MPT: the classical irreversible phosphorylation of the active site and an inhibition phase involving the reversible binding of MPT at a site peripheral to the active site, the peripheral organophosphorus site (POP-site). We now find that BW 284 C 51, a reversible specific inhibitor of AChE which protects the active site against irreversible inhibition by low MPT concentrations, can prevent the occurrence of the partially reversible inhibition phase. Hence, BW may bind to a peripheral site that either overlaps or is linked to the POP-site.
Acetylcholinesterase (ACHE; acetylcholine hydrolase; EC 3.1.1.7) is a widely distributed enzyme and is found in mammalian tissues, as well as in erythrocytes, skeletal muscle and nervous tissues (Hall, 1973; Rieger and Vigny, 1976; Ott and Brodbeck, 1978; Rieger et al., 1980; Massouli6 and Bon, 1982). The enzyme occurs in various molecular forms and is localized in several intra and extracellular sites (Younkin et al., 1982; Fernandez and Stiles, 1984; Dreyfus et al., 1985; G o u d o u et al., 1985). A potent uncharged phosphorylating c o m p o u n d (O-ethyl-S 2 diisopropylaminoethyl methyl phosphorothiolate; MPT) irreversibly inactivates A C h E (Vigny et al., 1978; G o u d o u et al., 1983), and also non-specific cholinesterases, at very low concentrations and in crude muscle extracts. We have previously shown that the kinetics of inhibition of mouse muscle A C h E and of each of its molecular forms do not follow pseudo-second order kinetics (Goudou et al., 1983). By using specific inhibitors of butyrylcholinesterase (BuChE; EC 3.1.1.8) and ACHE, we previously presented evidence that this behavior could not be explained by sequential inhibition of A C h E followed *Present address: Laboratoire de Technologie Enzymatique UA 523 CNRS, UTC, BP 233, 60206 Compiegne, France.
by BuChE. These results suggested an interaction of the M P T c o m p o u n d with a peripheral organophosphorus site (POP site) distinct from the active site on A C h E (Friboulet et al., 1983; G o u d o u et al., 1983; Friboulet et al., in preparation). BW 284 C 51 is a reversible bisquaternary a m m o n i u m specific inhibitor of A C h E and has a structure similar to that of several compounds known to act peripherally on ACHE. However, there is no report on any action of BW on a site other than the active site of ACHE. We present here evidence that such a peripheral interaction occurs between A C h E and the BW compound. We found that BW not only protects the A C h E active site against irreversible inhibition by M P T (Dreyfus et al., 1985; G o u d o u et al., 1985) but also interferes with the POP-site, dramatically changing the complex kinetic characteristics of M P T inhibition. EXPERIMENTAL PROCEDURES
Enzyme preparation Adult mice (129 ReJ strain) were sacrificed with ether and their sternocleidomastoid muscles were dissected out, rapidly washed in phosphate buffer saline (Ca 2+, Mg 2+ free), and weighed. The tissues were then homogenized in a conical glass to glass homogenizer, in I 0 volumes of a buffer containing 10raM Tris-HCl, pH7.0, 1M NaCI, l mM EGTA and I% Triton X-100. Homogenates were immediately centrifuged at 27,000g for 15 rain and supernatants
323
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324
were used for further experiments. AChE activity was assayed using the Ellman's method (Ellman et al., 1961). The artificial AChE films were prepared following a crosslinking with bovine serum albumin as a support (Thomas and Broun, 1976). An aliquot of 250 pl enzyme solution and 250~tl of a solution containing 10mg serum albumin, 1.25mg glutaraldehyde and 20raM phosphate buffer (pH 7.0) were mixed and gently spread on a planar glass surface. After 3 h, a complete insolubilization occurred. The resulting film was then rinsed and discs of I sq. cm area were stamped out and used for inhibition experiments.
hlhibition experiments Five to twenty mierolitres of concentrated aqueous solutions of the inhibitor were combined with a solution containing 0.1 M phosphate buffer (pH 7.0), 1.5mM DTNB and the free or insolubilized enzyme to give a final volume of 1 ml. Inhibitions were performed at 20C. The enzyme was exposed to various inhibitor concentrations for 45 min. After adding the substrate acetylthiocholine (1.5 mM final concentration), the remaining activities were spectrophotometrically measured at 412 nm at different times. In some experiments extensive washing of the remaining free inhibitor was performed (three times, 20min each with a solution containing phosphate buffer and DTNB) immediately before addition of the substrate. Protection oj AChE actitqty against inhibition by MPT hy B W 284 C 51 lnsolubilized AChE was preincubated with BW 284 C 51 at a fixed concentration for 45 min. The MPT compound was then added into the solution. After 45 min of incubation with the inhibitor, the preparations were washed twice, 45 rain each with a solution containing BW to avoid the reaction of free remaining MPT with the enzymatic sites previously protected by BW, and then washed twice in a solution free of BW containing phosphate buffer and DTNB. Control experiments were performed without adding the MPT inhibitor into the medium. Chemicals BW 284 C 51 (l,5-bis[4-allyldimethylammoniumphenyl]pentane-3-one dibromide) is a specific reversible inhibitor of AChE obtained from Burroughs Wellcome. MPT (O-ethyl SZ-diisopropylaminoethyl methyl phosphorothiolate) was a gift from Dr Leterrier (Hopital Percy, Paris, France). Acetylthiocholine iodide, 5:5' dithiobis-dinitrobenzoYc acid (DTNB), bovine serum albumin (fraction V) were obtained from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). All other chemicals used were of the highest available degree of purity. RESULTS
Reversible inhibition o f insolubilized muscle A C h E by B W 284 C 51 The insolubilization of A C h E by covalent binding into a proteic matrix allows a very rapid and efficient dialysis of the enzyme by simple washing. The enzyme is not d e n a t u r a t e d or kinetically altered by the insolubilization procedure as previously found in various kinetic studies (R6my et al., 1978; Friboulet and T h o m a s , 1982; G o u d o u et al., 1983; Friboulet et
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-log [BW] Fig. 1. Inhibition by BW284C 51 of insolubilized muscle ACHE. Inhibition of AChE activity as a function of BW concentration when the substrate was added after a 45 min incubation. (A) Soluble and ( 0 ) insolubilized ACHE. The bars indicate the SE of the mean for three independent determinations.
al., in preparation). The inhibition of insolubilized muscle A C h E was studied as a function of BW concentration, adding the substrate in the incubation m e d i u m after 45 rain exposure to the inhibitor (Fig. 1). We checked that the inhibition behavior was not different when A C h E was exposed to the inhibitor in solution instead of being insolubilized into a proteic matrix (Fig. 1). The inhibitory action of BW was fully reversible after extensive washing of insolubilized A C h E as shown by Fig. 2. N o irreversible phase of the reaction between A C h E and BW was detected. Competition between B W and M P T . / b r inhibition ()1 insolubilized muscle A ChE In a first series of experiments, the artificial films containing A C h E were exposed to M P T alone for 45 rain. The curve of residual activity as a function of M P T c o n c e n t r a t i o n did not follow a pseudo-second order kinetic as previously observed with the soluble enzyme and the isolated molecular forms (Fig. 3; see also G o u d o u et al., 1983), Moreover, when the artificial films were washed to eliminate any free inhibitor as described in Experimental Procedures, the inhibition curve showed an exaggerated d o m e shape, d e m o n s t r a t i n g a less efficient inhibition or a reversible inhibitory action of M P T in an M P T concentration range of a b o u t 5- 10 < 5 . 1 0 -8 M. In a n o t h e r series of experiments, insolubilized A C h E was preincubated for 45 min with BW 284 C 5 I, then incubated 45 min with different concentrations of M P T in the presence of BW and then washed four times for 45 rain each (twice in the presence of BW, then twice with p h o s p h a t e buffer and DTNB). The residual activity is shown on Fig. 4, plotted as a function of the M P T c o n c e n t r a t i o n for increasing BW concentrations. F o r low M P T concentrations,
A bisquaternary ammonium compound
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Fig. 2. Reversibility of the inhibition of insolubilized AChE by BW. Residual activity as a function of time of washing after a 45min incubation with BW (m) 10-8 M, (A) 10-7M, (l~) 10-6 M, (O) 10-SM or (O) 10 4 M. During the washing periods, the solutions were renewed every 15 min.
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-log [MPT] Fig. 3. Inhibition by MPT of insolubilized muscle ACHE. Residual activity as a function of MPT concentration during the inhibition period when the substrate was added in the incubation period (open symbols), or after elimination of free inhibitor (full symbols) by washing three times 20 min each. (A, A) Crude muscle homogenate ACHE, and (O, ©) 16 S isolated AChE molecular form. Bars: SE of the mean for three independent determinations.
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9 8 7 -log [MPT] Fig. 4. Inhibition by MPT of insolubilized muscle AChE in the presence of BW. Inhibition of AChE as a function of MPT concentration in the absence of BW ( 0 ) and when BW (O) 10-7M, ( . ) 10 6M, ([3) 10 -5 M or (A) 10 -4 M was present in the incubation medium. The substrate was added after 2 h washing to eliminate free MPT and bound BW.
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Fig. 5. Antagonist and agonist actions of the two inhibitors on muscle AChE activity. Protective effect of BW against inhibition of insolubilized AChE as a function of MPT concentration. A~w is the residual activity after inhibition by MPT in the presence of BW and Av in the absence of BW. BW concentrations were (C)) 10 7M, (11) 10 6M, (D) 10 5M and (A) 10 4M. the BW compound protects the A C h E activity against irreversible inhibition, as expected for an active site-directed inhibitor. However, for M P T concentrations higher than 10 9M, irreversible inhibition becomes almost total and the inhibition phase characterized by a less efficient M P T inhibition when M P T is used alone never appears. We also find that the inhibition resulting from the use of both inhibitors together is increased over the inhibition achieved with any one of the inhibitors used alone. There is an apparent synergistic action of the two inhibitors. This phenomenon is clearly shown by plotting ( A B w - A T ) / A T, where A~w stands for the residual A C h E activity after inhibition by M P T in the presence of BW and A v is the residual activity after inhibition by M P T in the absence of BW, against different BW concentrations (Fig. 5). DISCUSSION
We have evidence that the organophosphorus compound M P T not only acts on the active site but also on a peripheral regulatory site of mouse muscle A C h E (Goudou et al., 1983) that we called the peripheral organophosphorus site, POP-site (Friboulet et al., 1983; Friboulet et at., in preparation). M P T binding to the POP-site provokes a partial reversibility of the otherwise irreversible inhibition of A C h E which takes place at the esterasic active site. This partial reversal of inhibition only occurs for a narrow range of relatively high M P T concentrations (Fig. 3). In spite of its reversible characteristics at high M P T concentrations, nearly total irreversible inhibition of A C h E can be achieved at higher concentrations (better than 95% inhibition). It is well known
that several bisquaternary a m m o n i u m compounds (e.g. D-tubocurarine, succinylbischoline or decamethonium) bind to a site distinct from the active site of A C h E (Changeux, 1966: Wombacher and Wolf, 1971; Taylor and Lappi, 1975). The B W 2 8 4 C 5 1 compound is a potent bisquaternary reversible inhibitor of A C h E with no known peripheral action, although high concentrations of BW, in the micromolar range, exert a "curare-like" effect on endplate currents of frog muscle (Kordas et al., 1975) in a way similar to that observed with atropine and procaine, compounds which do interact peripherically on A C h E (Kato et al., 1972; Dawson and Poretski, 1984). When BW was used alone as an inhibitor or insolubilized muscle ACHE, the curves obtained were in agreement with a classical reversible inhibition or A C h E (Figs 1 and 2), without any irreversible phase of the reaction between BW and ACHE. When the protective action of BW against irreversible inhibition by M P T was studied, two phases could be observed. For M P T concentrations lower than 10 9 M, a protective action proportional to the BW concentration was observed (Figs 4 and 5). For higher concentrations of MPT, a synergy between the two inhibitors appeared. For higher BW concentrations, the final level of irreversible M P T inhibition was increased. This phenomenon occurs for M P T concentrations For which MPT interacts with the POP-site. We have previously shown that the insolubilization procedure is not responsible for unusual kinetics of inhibition of A C h E (Goudou et al., 1983; Fig. I). These results cannot be explained by the presence of low concentrations of BuChE or by the heterogeneity in the molecular forms since a preparation of isolated 16 S A C h E in which BuChE
A bisquaternary ammonium compound is negligible exhibits the same b e h a v i o r t h a n the p r e p a r a t i o n c o n t a i n i n g the total h o m o g e n a t e (Fig. 3). Moreover, recent results o b t a i n e d with single purified Torpedo californica A C h E forms show t h a t the pure enzyme also exhibits the u n u s u a l inhibition kinetics (Friboulet et al., in preparation). The total reversibility o f the inhibition of A C h E by BW and the " p r o t e c t i v e " effect of B W against low c o n c e n t r a t i o n s o f M P T , even at very high concentrations, eliminates the possibility of a n irreversible binding of B W on the active site or o n a peripheral site. These results can be fully explained if we assume that BW not only interacts reversibly with the A C h E active site a n d " p r o t e c t " it from irreversible inhibition by M P T , but also directly or indirectly reversibly interacts at higher c o n c e n t r a t i o n s with the POP-site. BW could exert its effect either by a direct binding on the POP-site or by blocking the M P T mediated induction o f a transient reversible M P T inhibition o f the active site after interaction with a peripheral site distinct from the POP-site. It is notew o r t h y that, when BW is used alone, there is n o deviation from classical reversible inhibition kinetics. This o b s e r v a t i o n suggests that either BW c a n n o t interact with the POP-site in the absence o f low c o n c e n t r a t i o n s of M P T , i.e. when the active site is occupied by M P T , or t h a t BW interaction with the POP-site does not result in a significant m o d u l a t i o n o f BW inhibitory characteristics at the active site. F u r t h e r kinetic studies in insolubilized conditions on the interactions between different classes of effectors acting on topographically distinct sites on the A C h E surface, such as the POP-site or the p r o p i d i u m site should open new routes in future a p p r o a c h e s to u n d e r s t a n d the p h a r m a c o l o g y of ACHE. Acknowledgements--This work was partially supported by the Direction des Recherches Etudes et Techniques and the Centre National de la Recherche Scientifique.
REFERENCES
Changeux J. P. (1966) Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs. Molec. Pharmac. 2, 369-392. Dawson R. M. and Poretski M. (1984) Procaine as a substrate and possible allosteric effector of cholinesterases. Neurochem. Int. 5, 559-569. Dreyfus P., Verdi6re M., Goudou D., Garcia L. and Rieger F. (1985) Acetylcholinesterase in mammalian skeletal muscle and sympathetic ganglion cells. Extra- and intracellular hydrophilic and hydrophobic asymmetric forms. In: Molecular Basis of Nerve Activity, (Changeux J. P., Hucho, F. and Neumann, E. eds), pp. 729-739. de Gruyter, Berlin.
327
Ellman G. L., Courtney K. D., Andres V. and Featherstone R. M. (1964) A new and rapid colorimetric determination of acetylcholinesterase. Biochem. Pharmac. 7, 88-95. Fernandez H. and Stiles J. R. (1984) Intra-versus extracellular recovery of 16S acetylcholinesterase following organophosphate inhibition in the rat. Neurosci. Lett. 49, 117-122. Friboulet A. and Thomas D. (1982) Electrical excitability of artificial enzyme membranes. Electrochemical and enzyme properties of immobilized acetylcholinesterase membranes. Biophys. Chem. 16, 145-151. Friboulet A., Goudou D. and Rieger F. (1983) Peripheral site of action of an organophosphorus compound, a methylphosphorothiolate derivative, on skeletal muscle acetylcholinesterase. Evidence for irreversible and reversible reactions on the extracted and immobilized enzyme. Abst. Second International Meeting on Cholinesterases (p. 132), Sept. 17 21 1983. Bled, Yugoslavia. Friboulet A., Rieger F., Goudou D., Amitai G. and Taylor P. Interaction of an organophosphorus compound with a peripheral site on acetylcholinesterase. In preparation. Goudou D., Friboulet A., Dreyfus P. and Rieger F. (1983) Ac~tylcholinest6rase et butyrylcholinest~rase dans le muscle squelettique de souris. Sp6cificit6 d'inhibition irr6versible par un compos6 organophosphor6 de type m&hylphosphorothiolate et formes mol6culaires multiples. C.r. hebd. Skanc. Acad. Sci., Paris 296, 169 172. Goudou D., Verdi6re M. and Rieger F. (1985) External and internal acetylcholinesterase in rat sympathetic neurones in vivo and in vitro. FEBS Lett. 186, 54-58. Hall Z. W. (1973) Multiple forms of acetylcholinesterase and their distribution in end-plate regions of rat diaphragm muscle. J. Neurobiol. 44, 343-361. Kato G., Tan E. and Yung J. (1972) Acetylcholinesterase. Kinetic studies on the mechanism of atropine inhibition. J. biol. Chem. 44, 3186-3189. Kordas M., Brzin M. and Majcen Z. (1975) A comparison of the effects of cholinesterase inhibitors on end-plate regions of rat diaphragm muscle. Neuropharmacology 14, 791-800. Massouli6 J. and Bon S. (1982) The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. A. Rev. Neurosci. 5, 57-I06. Ott P. and Brodbeck U. (1978) Multiple molecular forms of acetylcholinesterase from human erythrocyte membranes. Eur. J. Biochem. 88, 119 125. R6my M. H., David A. and Thomas D. (1978) Insolubilization and charge effects on crosslinked enzyme polymers. Kinetic studies in solution and in gelified membranes. FEBS Lett, 88, 332-336. Rieger F. and Vigny M. (1976) Solubilization and physicochemical characterization of rat brain acetylcholinesterase: development and maturation of its molecular forms. J. Neurochem. 27, 121 129. Rieger F., Ch6telat R., Nicolet M., Kamal L. and Poullet M. (1980) Presence of tailed asymmetric forms of acetylcholinesterase in the central nervous system of vertebrates. FEBS Lett. 121, 169-174. Taylor P. and Lappi S. (1975) Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding. Biochemistry 14, 1989-1997. Thomas D. and Broun G. (1976) Artificial enzyme membranes. Meth. Enzym. 44, 901-929.
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Vigny M., Bon S., Massouli6 J. and Leterrier F. (1978) Active site catalytic efficiency of acetylcholinesterase molecular forms in Electrophorus, Torpedo, rat and chicken. Eur. J. Biochem. 85, 317 323. Wombacher H. and Wolf H. U. (1971) Regulation of membrane-bound acetylcholinesterase activity by bis-
quaternary nitrogen compounds. Molec. Pharmac. 7, 554 566. Younkin S. G. Rosenstein C., Collins P. L. and Rosenberry T. L. (1982) Cellular localization of the molecular forms of acetylcholinesterase in rat. J. biol. Chem. 257, 13630 13637.
0197-0186/86 $3.00 +0.00 Pergamon Journals Ltd
INTERACTION OF A BISQUATERNARY A M M O N I U M C O M P O U N D WITH THE PERIPHERAL ORGANOPHOSPHORUS (POP) SITE ON ACETYLCHOLINESTERASE ALAIN FRIBOULET*, DANIf/LE GOUDOU and FRAN(~OIS RIEGER Unit6 de Biologie et Pathologie Neuromusculaires INSERM U 153, 17 rue du Fer-fi-Moulin, 75005 Paris, France (Received 8 August 1985; accepted 24 February 1986) Abstraet--O-ethyl-S (2 diisopropylaminoethyl) methyl phosphorothiolate (MPT) is an active site-directed inhibitor of acetylcholinesterase (ACHE). The inhibition of mouse muscle AChE by MPT as well as the inhibition of its individual molecular forms do not proceed as simple irreversible bimolecular reactions. The insolubilization of AChE into a semisolid matrix allows to characterize, after dialysis of all unbound ligand, a partially reversible phase of the inhibition by MPT. These results can be explained in terms of two different modes of inhibition by MPT: the classical irreversible phosphorylation of the active site and an inhibition phase involving the reversible binding of MPT at a site peripheral to the active site, the peripheral organophosphorus site (POP-site). We now find that BW 284 C 51, a reversible specific inhibitor of AChE which protects the active site against irreversible inhibition by low MPT concentrations, can prevent the occurrence of the partially reversible inhibition phase. Hence, BW may bind to a peripheral site that either overlaps or is linked to the POP-site.
Acetylcholinesterase (ACHE; acetylcholine hydrolase; EC 3.1.1.7) is a widely distributed enzyme and is found in mammalian tissues, as well as in erythrocytes, skeletal muscle and nervous tissues (Hall, 1973; Rieger and Vigny, 1976; Ott and Brodbeck, 1978; Rieger et al., 1980; Massouli6 and Bon, 1982). The enzyme occurs in various molecular forms and is localized in several intra and extracellular sites (Younkin et al., 1982; Fernandez and Stiles, 1984; Dreyfus et al., 1985; G o u d o u et al., 1985). A potent uncharged phosphorylating c o m p o u n d (O-ethyl-S 2 diisopropylaminoethyl methyl phosphorothiolate; MPT) irreversibly inactivates A C h E (Vigny et al., 1978; G o u d o u et al., 1983), and also non-specific cholinesterases, at very low concentrations and in crude muscle extracts. We have previously shown that the kinetics of inhibition of mouse muscle A C h E and of each of its molecular forms do not follow pseudo-second order kinetics (Goudou et al., 1983). By using specific inhibitors of butyrylcholinesterase (BuChE; EC 3.1.1.8) and ACHE, we previously presented evidence that this behavior could not be explained by sequential inhibition of A C h E followed *Present address: Laboratoire de Technologie Enzymatique UA 523 CNRS, UTC, BP 233, 60206 Compiegne, France.
by BuChE. These results suggested an interaction of the M P T c o m p o u n d with a peripheral organophosphorus site (POP site) distinct from the active site on A C h E (Friboulet et al., 1983; G o u d o u et al., 1983; Friboulet et al., in preparation). BW 284 C 51 is a reversible bisquaternary a m m o n i u m specific inhibitor of A C h E and has a structure similar to that of several compounds known to act peripherally on ACHE. However, there is no report on any action of BW on a site other than the active site of ACHE. We present here evidence that such a peripheral interaction occurs between A C h E and the BW compound. We found that BW not only protects the A C h E active site against irreversible inhibition by M P T (Dreyfus et al., 1985; G o u d o u et al., 1985) but also interferes with the POP-site, dramatically changing the complex kinetic characteristics of M P T inhibition. EXPERIMENTAL PROCEDURES
Enzyme preparation Adult mice (129 ReJ strain) were sacrificed with ether and their sternocleidomastoid muscles were dissected out, rapidly washed in phosphate buffer saline (Ca 2+, Mg 2+ free), and weighed. The tissues were then homogenized in a conical glass to glass homogenizer, in I 0 volumes of a buffer containing 10raM Tris-HCl, pH7.0, 1M NaCI, l mM EGTA and I% Triton X-100. Homogenates were immediately centrifuged at 27,000g for 15 rain and supernatants
323
ALAIN FRIBOULETet al,
324
were used for further experiments. AChE activity was assayed using the Ellman's method (Ellman et al., 1961). The artificial AChE films were prepared following a crosslinking with bovine serum albumin as a support (Thomas and Broun, 1976). An aliquot of 250 pl enzyme solution and 250~tl of a solution containing 10mg serum albumin, 1.25mg glutaraldehyde and 20raM phosphate buffer (pH 7.0) were mixed and gently spread on a planar glass surface. After 3 h, a complete insolubilization occurred. The resulting film was then rinsed and discs of I sq. cm area were stamped out and used for inhibition experiments.
hlhibition experiments Five to twenty mierolitres of concentrated aqueous solutions of the inhibitor were combined with a solution containing 0.1 M phosphate buffer (pH 7.0), 1.5mM DTNB and the free or insolubilized enzyme to give a final volume of 1 ml. Inhibitions were performed at 20C. The enzyme was exposed to various inhibitor concentrations for 45 min. After adding the substrate acetylthiocholine (1.5 mM final concentration), the remaining activities were spectrophotometrically measured at 412 nm at different times. In some experiments extensive washing of the remaining free inhibitor was performed (three times, 20min each with a solution containing phosphate buffer and DTNB) immediately before addition of the substrate. Protection oj AChE actitqty against inhibition by MPT hy B W 284 C 51 lnsolubilized AChE was preincubated with BW 284 C 51 at a fixed concentration for 45 min. The MPT compound was then added into the solution. After 45 min of incubation with the inhibitor, the preparations were washed twice, 45 rain each with a solution containing BW to avoid the reaction of free remaining MPT with the enzymatic sites previously protected by BW, and then washed twice in a solution free of BW containing phosphate buffer and DTNB. Control experiments were performed without adding the MPT inhibitor into the medium. Chemicals BW 284 C 51 (l,5-bis[4-allyldimethylammoniumphenyl]pentane-3-one dibromide) is a specific reversible inhibitor of AChE obtained from Burroughs Wellcome. MPT (O-ethyl SZ-diisopropylaminoethyl methyl phosphorothiolate) was a gift from Dr Leterrier (Hopital Percy, Paris, France). Acetylthiocholine iodide, 5:5' dithiobis-dinitrobenzoYc acid (DTNB), bovine serum albumin (fraction V) were obtained from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). All other chemicals used were of the highest available degree of purity. RESULTS
Reversible inhibition o f insolubilized muscle A C h E by B W 284 C 51 The insolubilization of A C h E by covalent binding into a proteic matrix allows a very rapid and efficient dialysis of the enzyme by simple washing. The enzyme is not d e n a t u r a t e d or kinetically altered by the insolubilization procedure as previously found in various kinetic studies (R6my et al., 1978; Friboulet and T h o m a s , 1982; G o u d o u et al., 1983; Friboulet et
100
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-log [BW] Fig. 1. Inhibition by BW284C 51 of insolubilized muscle ACHE. Inhibition of AChE activity as a function of BW concentration when the substrate was added after a 45 min incubation. (A) Soluble and ( 0 ) insolubilized ACHE. The bars indicate the SE of the mean for three independent determinations.
al., in preparation). The inhibition of insolubilized muscle A C h E was studied as a function of BW concentration, adding the substrate in the incubation m e d i u m after 45 rain exposure to the inhibitor (Fig. 1). We checked that the inhibition behavior was not different when A C h E was exposed to the inhibitor in solution instead of being insolubilized into a proteic matrix (Fig. 1). The inhibitory action of BW was fully reversible after extensive washing of insolubilized A C h E as shown by Fig. 2. N o irreversible phase of the reaction between A C h E and BW was detected. Competition between B W and M P T . / b r inhibition ()1 insolubilized muscle A ChE In a first series of experiments, the artificial films containing A C h E were exposed to M P T alone for 45 rain. The curve of residual activity as a function of M P T c o n c e n t r a t i o n did not follow a pseudo-second order kinetic as previously observed with the soluble enzyme and the isolated molecular forms (Fig. 3; see also G o u d o u et al., 1983), Moreover, when the artificial films were washed to eliminate any free inhibitor as described in Experimental Procedures, the inhibition curve showed an exaggerated d o m e shape, d e m o n s t r a t i n g a less efficient inhibition or a reversible inhibitory action of M P T in an M P T concentration range of a b o u t 5- 10 < 5 . 1 0 -8 M. In a n o t h e r series of experiments, insolubilized A C h E was preincubated for 45 min with BW 284 C 5 I, then incubated 45 min with different concentrations of M P T in the presence of BW and then washed four times for 45 rain each (twice in the presence of BW, then twice with p h o s p h a t e buffer and DTNB). The residual activity is shown on Fig. 4, plotted as a function of the M P T c o n c e n t r a t i o n for increasing BW concentrations. F o r low M P T concentrations,
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90 Time (rain)
180
Fig. 2. Reversibility of the inhibition of insolubilized AChE by BW. Residual activity as a function of time of washing after a 45min incubation with BW (m) 10-8 M, (A) 10-7M, (l~) 10-6 M, (O) 10-SM or (O) 10 4 M. During the washing periods, the solutions were renewed every 15 min.
100 1
I
I
10
I
9
I
1
8
7
-log [MPT] Fig. 3. Inhibition by MPT of insolubilized muscle ACHE. Residual activity as a function of MPT concentration during the inhibition period when the substrate was added in the incubation period (open symbols), or after elimination of free inhibitor (full symbols) by washing three times 20 min each. (A, A) Crude muscle homogenate ACHE, and (O, ©) 16 S isolated AChE molecular form. Bars: SE of the mean for three independent determinations.
501 O
L
11
\ 1
10
I
I
•
•--•
I
9 8 7 -log [MPT] Fig. 4. Inhibition by MPT of insolubilized muscle AChE in the presence of BW. Inhibition of AChE as a function of MPT concentration in the absence of BW ( 0 ) and when BW (O) 10-7M, ( . ) 10 6M, ([3) 10 -5 M or (A) 10 -4 M was present in the incubation medium. The substrate was added after 2 h washing to eliminate free MPT and bound BW.
326
ALAIN FRIBOULET eta/. 1
--
~#o
m----~'~
\ -1
A--l--~, 11
I 10
I 9
L 8
i 7
-log [MPT]
Fig. 5. Antagonist and agonist actions of the two inhibitors on muscle AChE activity. Protective effect of BW against inhibition of insolubilized AChE as a function of MPT concentration. A~w is the residual activity after inhibition by MPT in the presence of BW and Av in the absence of BW. BW concentrations were (C)) 10 7M, (11) 10 6M, (D) 10 5M and (A) 10 4M. the BW compound protects the A C h E activity against irreversible inhibition, as expected for an active site-directed inhibitor. However, for M P T concentrations higher than 10 9M, irreversible inhibition becomes almost total and the inhibition phase characterized by a less efficient M P T inhibition when M P T is used alone never appears. We also find that the inhibition resulting from the use of both inhibitors together is increased over the inhibition achieved with any one of the inhibitors used alone. There is an apparent synergistic action of the two inhibitors. This phenomenon is clearly shown by plotting ( A B w - A T ) / A T, where A~w stands for the residual A C h E activity after inhibition by M P T in the presence of BW and A v is the residual activity after inhibition by M P T in the absence of BW, against different BW concentrations (Fig. 5). DISCUSSION
We have evidence that the organophosphorus compound M P T not only acts on the active site but also on a peripheral regulatory site of mouse muscle A C h E (Goudou et al., 1983) that we called the peripheral organophosphorus site, POP-site (Friboulet et al., 1983; Friboulet et at., in preparation). M P T binding to the POP-site provokes a partial reversibility of the otherwise irreversible inhibition of A C h E which takes place at the esterasic active site. This partial reversal of inhibition only occurs for a narrow range of relatively high M P T concentrations (Fig. 3). In spite of its reversible characteristics at high M P T concentrations, nearly total irreversible inhibition of A C h E can be achieved at higher concentrations (better than 95% inhibition). It is well known
that several bisquaternary a m m o n i u m compounds (e.g. D-tubocurarine, succinylbischoline or decamethonium) bind to a site distinct from the active site of A C h E (Changeux, 1966: Wombacher and Wolf, 1971; Taylor and Lappi, 1975). The B W 2 8 4 C 5 1 compound is a potent bisquaternary reversible inhibitor of A C h E with no known peripheral action, although high concentrations of BW, in the micromolar range, exert a "curare-like" effect on endplate currents of frog muscle (Kordas et al., 1975) in a way similar to that observed with atropine and procaine, compounds which do interact peripherically on A C h E (Kato et al., 1972; Dawson and Poretski, 1984). When BW was used alone as an inhibitor or insolubilized muscle ACHE, the curves obtained were in agreement with a classical reversible inhibition or A C h E (Figs 1 and 2), without any irreversible phase of the reaction between BW and ACHE. When the protective action of BW against irreversible inhibition by M P T was studied, two phases could be observed. For M P T concentrations lower than 10 9 M, a protective action proportional to the BW concentration was observed (Figs 4 and 5). For higher concentrations of MPT, a synergy between the two inhibitors appeared. For higher BW concentrations, the final level of irreversible M P T inhibition was increased. This phenomenon occurs for M P T concentrations For which MPT interacts with the POP-site. We have previously shown that the insolubilization procedure is not responsible for unusual kinetics of inhibition of A C h E (Goudou et al., 1983; Fig. I). These results cannot be explained by the presence of low concentrations of BuChE or by the heterogeneity in the molecular forms since a preparation of isolated 16 S A C h E in which BuChE
A bisquaternary ammonium compound is negligible exhibits the same b e h a v i o r t h a n the p r e p a r a t i o n c o n t a i n i n g the total h o m o g e n a t e (Fig. 3). Moreover, recent results o b t a i n e d with single purified Torpedo californica A C h E forms show t h a t the pure enzyme also exhibits the u n u s u a l inhibition kinetics (Friboulet et al., in preparation). The total reversibility o f the inhibition of A C h E by BW and the " p r o t e c t i v e " effect of B W against low c o n c e n t r a t i o n s o f M P T , even at very high concentrations, eliminates the possibility of a n irreversible binding of B W on the active site or o n a peripheral site. These results can be fully explained if we assume that BW not only interacts reversibly with the A C h E active site a n d " p r o t e c t " it from irreversible inhibition by M P T , but also directly or indirectly reversibly interacts at higher c o n c e n t r a t i o n s with the POP-site. BW could exert its effect either by a direct binding on the POP-site or by blocking the M P T mediated induction o f a transient reversible M P T inhibition o f the active site after interaction with a peripheral site distinct from the POP-site. It is notew o r t h y that, when BW is used alone, there is n o deviation from classical reversible inhibition kinetics. This o b s e r v a t i o n suggests that either BW c a n n o t interact with the POP-site in the absence o f low c o n c e n t r a t i o n s of M P T , i.e. when the active site is occupied by M P T , or t h a t BW interaction with the POP-site does not result in a significant m o d u l a t i o n o f BW inhibitory characteristics at the active site. F u r t h e r kinetic studies in insolubilized conditions on the interactions between different classes of effectors acting on topographically distinct sites on the A C h E surface, such as the POP-site or the p r o p i d i u m site should open new routes in future a p p r o a c h e s to u n d e r s t a n d the p h a r m a c o l o g y of ACHE. Acknowledgements--This work was partially supported by the Direction des Recherches Etudes et Techniques and the Centre National de la Recherche Scientifique.
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